Objective: Gene expression analysis is widely used in most of studies of transcriptomic.Whether the cryopreserved samples storage time may influence the expression levels of genes is unknown.We aim to test the status of gene expression of different sample storage time during the cryopreserved processing by RNA sequencing.Methods: We selected one tissue sample of bladder cancer to be cryopreserved.Firsdy the sample was prepared to cell suspension divided into five centrifuge tube.One of five was undergone RNA sequencing processing immediately.The rest of them were storage in-80 ℃ by temperature gradient cooling method,and performed RNA sequencing after 18 h,66 h,10 d,30 d of storage.Then,we calculated the RPKM and FPKM of all mapped genes and those of 12 housekeeping genes.Results: Less than 30 d tissue storage had tittle effect on the total gene expression but induced changes in the transcript levels of stress-responsive genes as COX family after 18 h.Among the house keeping genes tested,the chosen 12 genes is stable.Conclusions: This study shows that the bladder cancer sample storage in-80 ℃ can be used in related research refer to RNA sequencing.The most genes expressions are stable except several stress-responsive genes.